The model organism C. elegans has many features that make it ideal for mapping functional elements encoded in its genome. We propose to build a genome-wide map of the binding sites for every C. elegans transcription factor. Moreover, over the course of this project, we will identify the exact localization of every transcription factor at single-cell resolution. These studies will be critical for deciphering the regulatory information housed in the genome to direct development and homeostasis. To maximize quality, speed, and comprehensiveness of the data collection, we have assembled a highly qualified team with extensive experience in performing the proposed goals, and developed a streamlined procedure for implementing these goals. First, we will make constructs in which each transcription factor is fused to a dual Myc-mCherry fluorescent tag. These constructs will be used to generate transgenic strains that express each tagged transcription factor at endogenous levels and locations. We will examine the temporal and spatial expression of each factor and determine its precise location using automated imaging techniques. Based on the optimal expression of each factor, we will perform genome-wide mapping of its binding sites in vivo using chromatin immunoprecipitation (ChIP) followed by hybridization to a microarray containing probes distributed across the entire genome. These binding data will be validated by multiple methods, including quantitative PCR of ChIP samples, expression analysis in transcription factor knockout strains, and mutational analysis of candidate binding sites in transgenic reporters. We will perform bioinformatic analysis to identify regulatory motifs and compare motifs between factors. All data generated from this project will be stored in a web-accessible in-house database for use by the scientific community, and incorporated into the modENCODE consortium database and Wormbase.